The present invention relates to novel interspecific Nicotiana excelsiorxc3x97N. benthamiana hybrid seeds and plants and to a method of producing interspecific Nicotiana hybrids having enhanced properties for biomass and the production of recombinant proteins using a viral vector system.
The difference between intra- and interspecific hybrids can be visualized by the Mendelian behavior of intraspecific hybrids and the essentially non-Mendelian behavior of interspecific hybrids. This difference results from the complete chromosome homology in hybrids between plants of the same species (intraspecific) and the partial or nonchromosome homology that characterizes hybrids between plants of different species (interspecific). The intermediate or gray area between the extremes among interspecific hybrids is seen in the behavior of those involving closely related species. Such species will have the same chromosome number, and the extent of chromosome homologies may be very high. Hybrids of this kind produce seed when self-pollinated and show evidence of Mendelian patterns of segregation for some traits.
Cytological studies of meiosis in some F1 hybrids may show evidence of chromosome irregularities that reflect the chromosomal differences that mark the parents as different species. A hybrid between distantly related species may show reduced pairing between chromosomes of the different genomes. Meiosis in such hybrids may exhibit the typical chromosome behavior characteristic of monogenomic haploids. Most interspecific F1 hybrid combinations in the genus Nicotiana fall into this latter category. Their chromosome doubled counterparts are called amphidiploids, or simply allopolyploids, when a parent of the hybrid may itself be a combination of different ancestral genomes. N. tabacum represents a typical example.
The relative difficulty of producing interspecific F1 hybrids increases in proportion to the decrease in taxonomic relations between their parents. Although some F1 hybrids are stable when converted to fertile polyploids, even hybrids between distantly related parents share some degree of chromosome homology. There are advantages to maintaining seed stocks of allopolyploids by self-pollination, particularly if the hybrid is difficult to obtain by conventional cross-pollination.
Interspecific hybridization and introgression in natural populations of plants and animals is a known source of genetic variation and adaptation. The term introgression, or introgressive transfer, of genetic traits is used in a narrower sense with short-term breeding projects. The goal of experimental interspecific hybridization is usually the introgressive transfer of a specific trait from one species (the nonrecurrent parent) into the genome of another (the recurrent parent). The trait must be expressed with reasonable phenotypic fidelity as a dominant or partial dominant in the F1 hybrid and all subsequent backcross generations, when nonrecurrent chromosomes are lost at random, otherwise the proposed interspecific transfer could not be made. Foreign genes from one species can become unstable when translocated into the germplasm of another.
Chromosome pairing between the foreign genomes in an interspecific hybrid may be extensive or minimal although the F1 hybrid is usually sterile. Fertility may be restored by treating germinating seed of the hybrid in 0.4 percent aqueous colchicine for about 4 hours to induce chromosome doubling. Immersion of the seeds in 0.1 percent colchicine for 24 hours may also be effective. Thereafter, the seed is rinsed in sterile distilled water and planted by sowing on the surface of pasteurized soil in glass or plastic preparation dishes. The seedlings later are transplanted to pots of soil in the greenhouse and mature plants that show good pollen development are self pollinated or backcrossed to the recurrent parent.
Three conventional approaches to interspecific hybridization for the purpose of alien transfer and incorporation of germplasm into stable diploid lines are (a) diploidxc3x97diploid then doubling to produce the allopolyploid and backcrossing to produce the sesquidiploid, (b) autotetraploidxc3x97autotetraploid to produce the allopolyploid directly, and (copyright) autotetraploidxc3x97diploid to produce the sesquidiploid directly as described in Technical Bulletin 1586, U.S. Department of Agriculture, 1979.
Gene transfer mediated by Agrobacterium tumefaciens vectors has become routine in tobacco (DeBlock et al., 1987; Grierson, et al., 1990; Hilder, et al., 1990; Lindbo and Dougherty, 1992). Commercial use of genetic transformation in agriculture depends on the incorporation of foreign genes into high-yielding germplasm. Plant breeders may want to combine several foreign genes into a single elite germplasm source. In order to accomplish this goal, designer chromosome construction is desirable.
Designer, or artificial, chromosomes have been produced in yeast. In plants, the scaffold for designer chromosome construction can be found in a breeding line that possesses the full complement of chromosomes from its own species plus an additional chromosome from a related species, Campbell, et al., Construction of a designer chromosome in tobacco, Theor Appl Genet (1994) 87:837-842. Additional chromosomes are often meiotically stable as homozygotes, and because recombination between additional chromosomes and the rest of the plant genome is rare (Gerstel 1945) the integrity of a foreign-gene linkage package can be preserved.
The placement of this linkage package on an additional chromosome will also minimize disturbance to the rest of the plant genome. The genetic structure of high-yielding germ plasm can be disturbed either through insertional mutagenesis or the disturbance of beneficial linkage blocks. Insertional mutagenesis, caused by the integration of foreign genes into plant coding sequences, can occur frequently. Konez, et al. (1989) estimated that at least 30% of all T-DNA insertions occur in transcribed regions of the Arabidopsis and Nicotiana genomes. Beneficial linkage blocks are formed through intermating and recombination followed by selection.
The desired traits for a plant may not be present in the germplasm of the species of interest. In such a case, traditional breeding within the species may not give acceptable results. Introducing a desired trait from one species into a related one by interspecific hybridization is followed by introgression into the recurrent parent.
Methods for expressing genes in plants has been described in U.S. Pat. Nos. 5,316,931; 5,589,367; 5,811,653 and 5,866,785, all of which are incorporated herein. Certain species have either an acceptable biomass or an acceptable expression of a sequence inserted into a plant viral vector, but no current species have both high levels of biomass and high levels of viral vector performance. There is a need for a method to develop in one species both the characteristics of increased biomass and improved viral vector performance.
The present invention is directed to a method of crossing two different species of Nicotiana to produce an interspecific hybrid which results in an increased biomass and improved host susceptibility to a viral vector.
In accordance with this invention, interspecific Nicotiana hybrids have been produced that are characterized with having a biomass of greater than that usually obtained from tobacco with CP-fusion constructs and a virion yield of greater than 1.0 mg. virion/gram fresh weight.
In one embodiment of the invention, there is provided a novel Nicotiana interspecific hybrid. This invention thus relates to the seeds of Nicotiana interspecific hybrid, to the plants of Nicotiana interspecific hybrid and to methods for producing a Nicotiana interspecific hybrid plant produced by crossing the Nicotiana interspecific hybrid with itself or another Nicotiana interspecific hybrid line.
Thus, any such methods using the Nicotiana interspecific hybrid are part of this invention: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using a Nicotiana interspecific hybrid as a parent are within the scope of this invention. Advantageously, the Nicotiana interspecific hybrid could be used in crosses with other, different, Nicotiana interspecific hybrids to produce first generation (F1) Nicotiana interspecific hybrid seeds and plants with superior characteristics.
In another aspect, the present invention provides for single gene converted plants of Nicotiana interspecific hybrid. The single transferred gene may preferably be a dominant or recessive allele. Preferably, the single transferred gene will confer such traits as herbicide resistance, insect resistance, resistance for bacterial, fungal, or viral disease, male fertility, male sterility, enhanced nutritional quality, and industrial usage. The single gene may be a naturally occurring Nicotiana gene or a transgene introduced through genetic engineering techniques.
In another aspect, the present invention provides regenerable cells for use in tissue culture of a Nicotiana interspecific hybrid. The tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing Nicotiana interspecific hybrid, and of regenerating plants having substantially the same genotype as the foregoing Nicotiana interspecific hybrid. Preferably, the regenerable cells in such tissue cultures will be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods or stems. Still further, the present invention provides Nicotiana plants regenerated from the tissue cultures of the invention.
To provide an understanding of several of the terms used in the specification and claims, the following definitions are provided:
Geneware(trademark) viral vectorxe2x80x94As used herein, Geneware(trademark) viral vector refers to a system for expressing genes in plants, such as described in U.S. Pat. Nos. 5,316,931; 5,589,367; 5,811,653 and 5,866,785, all incorporated herein by reference.
Biomassxe2x80x94As used herein, the term xe2x80x9cbiomassxe2x80x9d means the total fresh weight in rams of plant tissue which is harvested above the ground. The terms xe2x80x9cfresh weightxe2x80x9d and xe2x80x9cbiomassxe2x80x9d are used interchangeably herein.
Virion yieldxe2x80x94As used herein the term xe2x80x9cvirion yieldxe2x80x9d means the total yield of the virus measured in mg of virion per gram fresh weight.
Hostxe2x80x94As used herein, a xe2x80x9chostxe2x80x9d means a cell, tissue or organism capable of supporting replication of a vector or plant viral nucleic acid and which is capable of being infected by a virus containing the viral vector or plant viral nucleic acid.
Infectionxe2x80x94As used herein, an xe2x80x9cinfectionxe2x80x9d means the ability of a virus to transfer its nucleic acid to a host or introduce viral nucleic acid into a host, wherein the viral nucleic acid is replicated, viral proteins are synthesized, and new viral particles assembled. In this context, the terms xe2x80x9ctransmissiblexe2x80x9d and xe2x80x9cinfectivexe2x80x9d are used interchangeably herein.
Phenotypic Traitxe2x80x94As used herein, xe2x80x9cphenotypic traitxe2x80x9d is an observable property resulting from the expression of a gene or genes.
Plant Tissuexe2x80x94As used herein, xe2x80x9cplant tissuexe2x80x9d is any tissue of a plant in planting or in culture. This term is intended to include a whole plant, plant cell, plant organ, protoplast, cell culture, or any group of plant cells organized into a structural and functional unit.
Recombinant Plant Viral Nucleic Acidxe2x80x94As used herein, xe2x80x9crecombinant plant viral nucleic acidxe2x80x9d means it has been modified to contain nonnative nucleic acid sequences.
Recombinant Plant Virusxe2x80x94As used herein, xe2x80x9crecombinant plant virusxe2x80x9d is a plant virus containing the recombinant plant viral nucleic acid.
Vectorxe2x80x94As used herein, xe2x80x9cvectorxe2x80x9d is a self-replicating nucleic acid molecule which transfers a nucleic acid segment between cells.
Virusxe2x80x94As used herein, xe2x80x9cvirusxe2x80x9d is an infectious agent composed of a nucleic acid encapsidated in protein. A virus may be a mono-, di-, tri- or multi-partite virus.
Backcrossingxe2x80x94As used herein, xe2x80x9cbackcrossingxe2x80x9d is a process in which a breeder repeatedly crosses hybrid progeny back to one of the parents, for example, a first generation hybrid F1 with one of the parental genotypes of the F1 hybrid.
Essentially all the physiological and morphological characteristicsxe2x80x94As used herein, a plant having essentially all the physiological and morphological characteristics means a plant having the physiological and morphological characteristics, except for the characteristics derived from the converted gene.
Single Gene Converted (Conversion)xe2x80x94As used herein, xe2x80x9csingle gene converted (conversion)xe2x80x9d plant refers to plants which are developed wherein essentially all of the desired morphological and physiological characteristics of a variety are recovered in addition to the single gene transferred into the variety via the backcrossing technique or via genetic engineering.
Trichomesxe2x80x94As used herein, xe2x80x9ctrichomesxe2x80x9d means the glandular hairs on the surface of the plant stem, leaf, calyx, corolla or other plant tissue surfaces.
Leaf laminaxe2x80x94As used herein, xe2x80x9cleaf laminaxe2x80x9d means the leaf surface devoid of vascular tissue of primary, secondary or ternary nature.
GFP-GJxe2x80x94As used herein, xe2x80x9cGFP-GJxe2x80x9d means a crude plant extract containing green fluorescent protein.
GFP-pH5, xcex94xe2x80x94As used herein, xe2x80x9cGFP-pH5, xcex94xe2x80x9d means a crude plant extract that was treated with heat and a pH of 5 that contains green fluorescent protein that is enriched for its presence.
CP-GJxe2x80x94As used herein, xe2x80x9cCP-GJxe2x80x9d means a crude plant extract containing viral coat protein.
CP-pH5, xcex94xe2x80x94As used herein, xe2x80x9cCP-pH5, xcex94xe2x80x9d means a crude plant extract that was treated with heat and a pH of 5 that contains coat protein that is enriched for its presence.
Host plant species vary in their ability to support expression of a sequence inserted into a plant viral vector. For example, some species (such as Nicotiana benthamiana) support expression from a dual-subgenomic promoter tobamoviral vector to a high specific activity, but have relatively low biomass. Other species (such as N. tabacum) have high biomass and/or other desirable properties for growth in the field, but have a relatively low specific activity of the expressed protein. Using the method of the present invention, after chromosome doubling to restore fertility, the primary hybrid may have suitable properties, or it may be desirable to backcross toward either parent selecting or screening at each generation for the desired properties of the non-recurrent parent (e.g., introgressing the superior biomass of N. tabacum into N. benthamiana, or introgressing the superior viral vector performance of N. benthamiana into N. tabacum). A viral vector expressing the green fluorescent protein (GFP) is one example of a useful tool for screening the level of systemic expression in candidate hybrid plants. Geneware(trademark) viral vector refers to a system for expressing genes in plants such as described in U.S. Pat. Nos. 5,316,931; 5,589,367; 5,811,653 and 5,866,785, all incorporated herein by reference.